8 research outputs found
Saccharides as Prospective Immobilizers of Nucleic Acids for Room-Temperature Structural EPR Studies
Pulsed dipolar electron paramagnetic
resonance (EPR) spectroscopy
is a powerful tool for structural studies of biomolecules and their
complexes. This method, whose applicability has been recently extended
to room temperatures, requires immobilization of the studied biosystem
to prevent averaging of dipolar couplings; at the same time, the modification
of native conformations by immobilization must be avoided. In this
work, we provide first demonstration of room-temperature EPR distance
measurements in nucleic acids using saccharides trehalose, sucrose,
and glucose as immobilizing media. We propose an approach that keeps
structural conformation and unity of immobilized double-stranded DNA.
Remarkably, room-temperature electron spin dephasing time of triarylmethyl-labeled
DNA in trehalose is noticeably longer compared to previously used
immobilizers, thus providing a broader range of available distances.
Therefore, saccharides, and especially trehalose, can be efficiently
used as immobilizers of nucleic acids, mimicking native conditions
and allowing wide range of structural EPR studies at room temperatures
A Versatile Approach to Attachment of Triarylmethyl Labels to DNA for Nanoscale Structural EPR Studies at Physiological Temperatures
Triarylmethyl
(trityl, TAM) radicals are a promising class of spin
labels for nanometer-scale distance measurements in biomolecules at
physiological temperatures. However, to date, existing approaches
to site-directed TAM labeling of DNA have been limited to label attachment
at the termini of oligonucleotides, thus hindering a majority of demanded
applications. Herein, we report a new versatile strategy for TAM attachment
at arbitrary sites of nucleic acids. It utilizes an achiral non-nucleoside
phosphoramidite monomer for automated solid-phase synthesis of oligonucleotides,
which are then postsynthetically functionalized with TAM. We demonstrate
a synthesis of a set of oligonucleotide complexes that are TAM-labeled
at internal or terminal sites, as well as the possibility of measuring
interspin distances up to ∼5–6 nm at 298 K using double
quantum coherence electron paramagnetic resonance (EPR). Implementation
of the developed approach strongly broadens the scope of nucleic acids
and nucleoprotein complexes available for nanoscale structural EPR
studies at room temperatures
Triarylmethyl Labels: Toward Improving the Accuracy of EPR Nanoscale Distance Measurements in DNAs
Triarylmethyl (trityl, TAM) based
spin labels represent a promising
alternative to nitroxides for EPR distance measurements in biomolecules.
Herewith, we report synthesis and comparative study of series of model
DNA duplexes, 5′-spin-labeled with TAMs and nitroxides. We
have found that the accuracy (width) of distance distributions obtained
by double electron–electron resonance (DEER/PELDOR) strongly
depends on the type of radical. Replacement of both nitroxides by
TAMs in the same spin-labeled duplex allows narrowing of the distance
distributions by a factor of 3. Replacement of one nitroxide by TAM
(orthogonal labeling) leads to a less pronounced narrowing but at
the same time gains sensitivity in DEER experiment due to efficient
pumping on the narrow EPR line of TAM. Distance distributions in nitroxide/nitroxide
pairs are influenced by the structure of the linker: the use of a
short amine-based linker improves the accuracy by a factor of 2. At
the same time, a negligible dependence on the linker length is found
for the distribution width in TAM/TAM pairs. Molecular dynamics calculations
indicate greater conformational disorder of nitroxide labels compared
to TAM ones, thus rationalizing the experimentally observed trends.
Thereby, we conclude that double spin-labeling using TAMs allows obtaining
narrower spin–spin distance distributions and potentially more
precise distances between labeling sites compared to traditional nitroxides
Physiological-Temperature Distance Measurement in Nucleic Acid using Triarylmethyl-Based Spin Labels and Pulsed Dipolar EPR Spectroscopy
Resolving
the nanometer-scale structure of biomolecules in natural
conditions still remains a challenging task. We report the first distance
measurement in nucleic acid at physiological temperature using electron
paramagnetic resonance (EPR). The model 10-mer DNA duplex has been
labeled with reactive forms of triarylmethyl radicals and then immobilized
on a sorbent in water solution and investigated by double quantum
coherence EPR. We succeeded in development of optimal triarylmethyl-based
labels, approach for site-directed spin labeling and efficient immobilization
procedure that, working together, allowed us to measure as long distances
as ∼4.6 nm with high accuracy at 310 K (37 °C)